WO 2004/080492 A1 discloses a method for radiofluorination of a biological targeting vector, comprising reaction of a compound of formula (I) with a compound of formula (II):
or,    a compound of formula (III) with a compound of formula (IV)
wherein:                R1 is an aldehyde moiety, a ketone moiety, a protected aldehyde such as an acetal, a protected ketone, such as a ketal, or a functionality, such as diol or N-terminal serine residue, which can be rapidly and efficiently oxidised to an aldehyde or ketone using an oxidising agent;        R2 is a group selected from primary amine, secondary amine, hydroxylamine, hydrazine, hydrazide, aminooxy, phenylhydrazine, semicarbazide, and thiosemicarbazide;        R3 is a group selected from primary amine, secondary amine, hydroxylamine, hydrazine, hydrazide, aminooxy, phenylhydrazine, semicarbazide, or thiosemicarbazide;        R4 is an aldehyde moiety, a ketone moiety, a protected aldehyde such as an acetal, a protected ketone, such as a ketal, or a functionality, such as diol or N-terminal serine residue, which can be rapidly and efficiently oxidised to an aldehyde or ketone using an oxidising agent;            to give a conjugate of formula (V) or (VI) respectively:
wherein X is —CO—NH—, —NH—, —O—, —NHCONH—, or —NHCSNH—, and is preferably —CO—NH—, —NH— or —O—; Y is H, alkyl or aryl substituents; and    the Linker group in the compounds of formulae (II), (IV), (V) and (VI) is selected from:
                wherein:        n is an integer of 0 to 20;        m is an integer of 1 to 10;        p is an integer of 0 or 1;        Z is O or S.        
Poethko et al [J. Nucl. Med., 45(5), 892-902 (2004)] disclose a method of radiolabelling peptides with the radioisotope 18F, wherein an aminooxy-functionalised peptide is condensed with [18F]-fluorobenzaldehyde to give a labelled peptide having an oxime ether linkage as follows:

Schottelius et al [Bioconj. Chem., 19(6), 1256-1268 (2008)] further developed the method of Poethko et al. Schottelius et al use an aminooxy-functionalised peptide wherein the amine of the aminooxy group is protected with an N-Boc (Boc=tert-butyloxycarbonyl) protecting group. The desired aminooxy-functionalised peptide is generated in situ in the presence of [18]-fluorobenzaldehyde via deprotection of the N-Boc group at acidic pH (pH=2) at 75° C. Schottelius et al used a 5-fold molar excess of the Boc-protected precursor, because the deprotection was not quantitative under the reaction conditions.
Mezo et al [J. Pept. Sci., 17, 39-46 (2010)] describe some of the problems associated with the above oxime ligation chemistry of Boc-protected aminooxy-functionalised peptides. Thus, it is known that the Boc-aminooxy reagent can acylate formed Boc-protected aminooxy-peptide, leading to undesirable by-products. It is also known that the reactivity of the free aminooxy group of the functionalised peptide is high towards carbonyl compounds. Consequently, unwanted condensation can occur with any adventitious aldehydes or ketones present either in the reaction mixture or in any subsequent purification steps. Such aldehydes or ketones could be traces of acetone present in the solvents used, or formaldehyde (e.g. from plasticizers). Mezo et al are interested in solving this problem for both the conjugation of anti-cancer drugs and of [18F]-fluorobenzaldehyde to peptides. Mezo et al solve the problem by carrying out the deprotection of the Boc-aminooxy peptide in the presence of a tenfold molar excess of free (aminooxy)acetic acid (Aoa) as a ‘carbonyl capture agent’. The deprotected aminooxy-peptide and excess Aoa is then lyophilised and stored at 4° C. Immediately prior to the oxime ligation reaction, the lyophilised mixture is reconstituted, and excess Aoa is separated by HPLC or Sep-Pak plus C18 cartridge. Mezo et al provide an example in which non-radioactive (i.e. 19F) 4-fluorobenzaldehyde is conjugated to an aminooxy-functionalised somatostatin peptide using this technique. Mezo et al do not provide any data on 18F-radiolabelling.
WO 2012/072736 discloses the use of alternative protecting group chemistry for the aminooxy groups of functionalised biomolecules. The protected aminooxy group is of formula:
                wherein R1 and R2 are independently chosen from C1-3 alkyl, C1-3 fluoroalkyl or C4-6 aryl.        
WO 2012/072736 teaches that it is preferred to deprotect the protected aminooxy group in situ, i.e. in the presence of the radioactive aldehyde to which it is to be conjugated without intermediate isolation. Example 3 of WO 2012/072736 does, however, disclose the deprotection of a peptide functionalised with such a protected aminooxy group, and subsequent direct lyophilisation to afford an isolated product. The lyophilised composition obtained directly was described as pure. WO 2012/072736 does not, however, recognise the problems of side reactions with acetate forming by-products as taught by the present invention.
There is therefore still a need for alternative or improved methods of preparing and radiolabelling peptides and other biological targeting molecules.
The Present Invention.
The present invention provides a method for preparing a lyophilised composition of a biological targeting molecule (BTM) having conjugated thereto an aminooxy functional group.
The invention provides purified aminooxy-functionalised BTMs as lyophilised compositions of such purified materials, and their subsequent use in radiolabelling. The invention provides a more efficient deprotection step due to increased solubilisation of the protected precursor. Since the deprotection occurs in the mobile phase used for chromatographic purification, the process is more efficient since there is no need to isolate the purified protected aminooxy derivative. This is particularly important since it saves a lyophilisation step, since the protected aminooxy derivative is typically isolated by lyophilisation which is a time-consuming process. Also, the lyophilised material is more readily dissolved when used for radiolabelling which is important for efficient labelling and purification of BTMs.